Abstract Symbiotic human gut bacteria perform a number of beneficial physiological processes, including fermentation of dietary fiber polysaccharides and instruction of mucosal immune development. During homeostasis, the immune system utilizes several mechanisms to tolerate gut microbes; however, the same symbionts can cause inflammation when certain defects in the immune system exist. Gut symbionts have also evolved mechanisms to protect themselves from the host immune response, including the propensity to synthesize a variety of different surface capsular polysaccharides (CPS). Bacteroides thetaiotaomicron (B. theta) is a model gut symbiont that degrades a wide variety of dietary, host, and microbial glycans and dedicates hundreds of genes to CPS production. The ability to express multiple different capsules, along with their complex, phase-variable regulation, is a common feature of gut Bacteroidales species, including B. fragilis and B. theta. One of the many B. fragilis CPS, termed PSA, is zwitterionic, and has been shown to modulate host cytokine levels and induce anti-inflammatory responses that limit intestinal disease. Recently, several other diverse intestinal bacteria, besides B. fragilis, have been shown to produce zwitterionic CPS that also exhibit anti-inflammatory properties. Thus, it has emerged that several CPS, among just a small set studied, have evolved bioactive properties. In parallel, genome sequencing has revealed a bewildering diversification of genetic loci involved in CPS by human gut bacteria. The central hypothesis of this proposal is that other gut bacterial CPS (especially non-zwitterionic forms) have evolved and been selected under immune pressure to possess a variety of immunomodulatory functions. To test this premise, we have created a novel experimental system: a defined CD4+ TCRtg mouse line in which T cells are specific for a B. theta outer membrane protein, combined with a set of B. theta strains that each either express a single CPS or none at all. Our hypothesis is supported by two major findings that set the basis for this proposal. First, when eight individual strains that each express a different B. theta CPS were tested for their ability to be processed and presented by macrophages to stimulate T cells in vitro, there is a broad range of activities, from weak to strong. Second, analysis of just 14 other sequenced B. theta strains, revealed extensive CPS diversification. Strikingly, we identified a total of 49 unique cps loci among just these 14 strains, suggesting that the ?universe? of Bacteroides CPS is large. We propose to test our hypothesis through two aims: 1) Determine the mechanisms by which 4 individual B. theta capsules exert their range of immunomodulatory activities, and 2) Isolate and express novel CPS genes from Bacteroides species and measure their immunomodulatory activity in vivo and in vitro. Polysaccharides are the most diverse class of biomolecules in nature and have the potential to exert potent biological effects. These studies will expand the known lexicon of bacterial polysaccharide-immune system interactions and should lead to discovery of new, bioactive CPS for potential use as therapeutics.